The annual influenza virus epidemics represent an important cause of morbidity and mortality throughout the world. In the United States of America it is estimated that more than 200,000 people are hospitalized each year for syndromes connected to influenza viruses, with about 40,000 deaths more or less directly related thereto (Thompson et al., JAMA, 2003, 289:179-186). Apart from these figures we must also consider the cases, in exponentially higher numbers, of infected subjects that stay at home for more or less long periods, with inevitable economic repercussions due to the loss of working days. A recent work (Molinari et al., Vaccine, 2007, 25: 5086-5096) has estimated the medical costs directly related to annual epidemics at 10.4 billions of US dollars per year, to which 16.3 billions of US dollars must be added for lost earnings due to absence from work. If in the calculation we consider other items too, such as the monetization of the economical losses linked to the death of the infected subjects, the amount rises to the incredible figure of 87.1 billions of US dollars. These economical data linked with the annual epidemics, together with a dreaded pandemic that could occur at any moment in the near future due to the appearance of influenza viruses new to man, explain the considerable interest in the search for effective strategies to contain the spread of these viruses.
Currently, the only available tool for facing the annual influenza epidemics in some way is an inactivated trivalent vaccine containing viral isolate antigens that presumably will be responsible for the epidemic of the next influenza season. This kind of prediction, based on epidemiological data linked to early isolations in some sentinel geographic areas, does not always turn out to be correct. Thus, there is a not at all negligible risk, which is present year after year, that the trivalent vaccine developed for a certain influenza season might prove substantially ineffective.
In that case, as well as in the case of a new pandemic, the only available prophylactic/therapeutic aid would be to resort to the two available classes of antiviral drugs: the M2 protein inhibitors (amantadine and rimantadine), and the neuraminidase inhibitors (oseltamivir and zanamivir). However, in this situation too, a series of problems can be already expected, related both to the need to administer the antivirals in a very early stage of the infection, and to the rapid appearance, which has already occurred however, of resistant viral isolates.
An alternative effective strategy could be based on neutralizing antibody preparations directed against critical viral proteins and capable of recognizing portions of such proteins which are shared among the different isolates of influenza viruses.
For better understanding of the potential of an approach based on the passive administration of antibodies, it is useful to briefly mention the main structural features of the influenza viruses. The influenza viruses belong to the Orthomyxoviridae family and are characterized by the presence of an envelope derived from infected cell membranes, on which approximately 500 spikes are present, also referred to as projections. Such projections consist of trimers and tetramers from two important viral surface proteins: hemagglutinin (HA) and neuraminidase (NA). An integral membrane protein (M2) is also found on the envelope surface, which protein is present in much lower numbers compared to hemagglutinin and neuraminidase, and also organized in tetramers.
The influenza virus is further characterized by the presence, within the core, of a segmented genome comprised of 8 single stranded RNA fragments. Based on the features of some proteins within the virion (NP and MD, three influenza virus types are recognizable: type A, type B, and type C.
Type A and type B viruses are responsible for the annual epidemics. Instead, type C viruses are responsible for less severe syndromes.
Within type A viruses (the only ones responsible for pandemics and capable of causing the most severe syndromes even during annual epidemics), different subtypes are also recognizable based on the antigenic features of hemagglutinin and neuraminidase. The subtypes that have affected humans in the course of recent history are subtypes H1N1 and H3N2 (still circulating at present and contained in vaccine preparations), as well as subtype H2N2, no longer circulating since 1968 and responsible for the so called “Asiatic” flu in 1957. Other subtypes have sporadically affected humans (H9N2, H7N7, and the so dreaded recent H5N1 subtype), but they have not succeeded in spreading effectively and displacing the circulating subtypes.
The role of the surface proteins is essential in the viral replication cycle. In particular, hemagglutinin is the protein that allows the virus to recognize the sialic acid present on the surface of some cells, and to infect them. Instead, neuraminidase operates at the end of the viral replication cycle, that is during the release of new virus particles from the infected cells. Its function is to assist the release of hemagglutinin of the newly formed virions from the sialic acid present on the surface of the cell that produced them. The key role played by these two proteins, as well as their display on the virus surface, explain why they represent the main target of the immune response, and why they are susceptible to a high rate of mutation. In fact, the annual epidemics are caused by viruses that are more or less different from the ones of the previous years, and therefore are more or less effectively able to escape the immune response they stimulated. In other words, the progressive accumulation of point mutations in hemagglutinin (mostly) and neuraminidase (secondarily) makes the protective antibodies, produced in the course of previous epidemics, on the whole progressively ineffective.
The main protective role within the anti-influenza immune response is played by the humoral component. Antibodies exert their protective role primarily interfering with the binding of hemagglutinin to sialic acid, thereby preventing infection of the cells. Such a selective pressure determines the high rate of mutation in hemagglutinin Sequence studies performed on H3 hemagglutinin subtype from 1968 through 1999 have revealed a total of 101 amino acid mutations (on a total of approximately 560 amino acids), with an average of about 3.5 mutations per year. 60% of mutations which occurred in the studied period were retained in the circulating viruses the following year too, indicative of the persistence of an immune-mediated selective pressure. 95% of these mutations were found in the HA1 hemagglutinin subunit, that is the one directly involved in the binding to sialic acid. Within such a high variability, however, some unchanged amino acid residues have been found, indicative of their essential role in the function of the protein. These hemagglutinin portions represent a potential target for a cross-neutralizing response towards the different subtypes of influenza viruses. However, it is predictable that such regions will not be able to induce an effective antibody response in most patients, since the fact that such targets hide in immunosilent areas has certainly represented a very favorable evolutionary step for the virus.
In fact, when referring to anti-influenza immunity, three different types of immunity must be taken into consideration, which can be well understood in the light of the data reported above
HOMOLOGOUS IMMUNITY: related to the individual isolate. This type of immunity is always seen after an infection or a vaccination, but it provides a very limited protection against other isolates.
HOMOSUBTYPE IMMUNITY: related to isolates belonging to the same subtype. This type of immunity is often seen after an infection or a vaccination, but is lost when the mutation rate in hemagglutinin and/or neuraminidase increases considerably.
HETEROSUBTYPE IMMUNITY: related to isolates belonging to different subtypes. This type of immunity is extremely rare both in case of natural infection and in case of vaccination. From the strategic point of view, it is the most important immunity, as its presence and stimulation would be equivalent to the resistance to infection by every type A influenza virus.
Until a few years ago, it was thought that the heterosubtype immunity could be achieved just by stimulating effectively cellular immunity components directed against less mutated viral proteins, such as for example the NP protein that constitutes its core. However, recent studies have shown that mice depleted of CD8 lymphocytes are still able to display a heterosubtype immunity, in contrast with mice depleted of the type B lymphocyte component (Nguyen H H, J Inf. Dis. 2001, 183: 368-376). An even more recent study has confirmed this data, highlighting a crucial role of antibodies, even if not neutralizing, directed precisely against epitopes that are conserved among the different subtypes (Rangel-Moreno et al. The J of Immunol, 2008, 180: 454-463).